Link 4-Melanin 95-97

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                                                     In memory of Giovanna Misuraca  

We do not arrive at ideas, they reach us. The courage to think comes down from the urgent desire to be, then destiny blooms. (MARTI N   HEIDEGGER)

Brown substances and black substances are very common  in the lithosphere, the hydrosphere, the atmosphere and the biosphere, and if one considers the black matter found in interstellar spaces also in the cosmos (  Link 7 and Link 8 ). The natural and synthetic black substances ( BCM and BSM ) of the biosphere are called melanins  (Link 22).Characteristic of the black material is conductivity / superconductivity binding of organic and inorganic products, storage of liquids and gases,assembling cellular factor. All the natural and synthetic black substances which are cited in the paper are characterised by a vast chromatic variation which, however, always goes through the colour range : black - vermillion - yellow.

Typical substances which present these chromatic variations are:

Sulphur of cadmium and other sulphurs (lithosphere) .

Humic acids (hydrosphere, lithosphere)

Skin and hair pigments (biosphere)

Pyrrole black (synthesis)

In the case of cadmium sulphur colour is linked to the threshold of conductibility. The lower the threshold the darker the colour. Pyrrole –black and acetylene-black are   the most studied organic semiconductors. The idea that natural melanin is also a semiconductor is not completely new . The works of the 1960s seem to have been neglected by the contemporary scientific community. S. T. Allen and D. J. E. Ingram say  : ... In conclusion, the free-radical property and the radiation properties of pigmented materials may be understood if it is supposed that the melanin  ( link 22) acts as a one dimensional semiconductor with bound protons producing traps in the system .... The study of conducibility of the biological melanins will be, we are certain, one of the next objectives of world scientific research. Besides, it should be stressed that the various melanogenesis in vitro and in vivo are fluctuating chemical systems, in the sense predicted and studied by Piccardi  (Florence) sensitive to variables of a terrestrial, solar and cosmic nature. Both the structure of the molecule and the colloidal structure is important in a radical process like that of melanin. In the text the term eumelanin , pheomelanin ,  allomelanin  are used to designate the black or red-brown substances of biological or not .Whereas natural pigments are called BCM ( black cell matter)  the synthetic is indicated with the monogram BSM  ( black synthetic matter ). It is hoped that in the future the scientific community finds new terms to indicate "blacks" of all origins in a single context. A brief aside before beginning the story. In the Museum of Fine Arts in Basel there are some tables of the altar of Heilospeigel by K. Wits, these are chromatically very different from one another and where they appear in full light, among the different colours are those of the semiconductors. The expressive meaning of the artist’s operative processes and his capacity of distributing colours are shown by fact that the more one thinks of the expressive value of the colours, the more they seem mysterious. An analyis of the expressive meaning of the colours of the semiconductors should be illustrated, in the future. In the mind of a painter the black (a dark grey colour) is obtained also from the mixing of a set of varnishes of different types and colours. For the painter the dark colours symbolise the strong negative of life while the light colours represent the positive side of life. Mourning is black, even though it is white and luminous in other populations. The black cat which crosses the road of a latino is an ill omen but in anglo-saxon populations it is a sign of good luck. The study of chemists and physicists on melanins in this century have not always lead to clarifications, but rather to taking up positions or points of view which are not only discordant among themselves but also in disagreement with the classical philosophy of the chemical science of natural organic substances.

Chemistry and evolution.

There are essentially two types of mechanism which transform the black substances, starting from very simple composites of carbon, subjected to natural forces like temperature, electrical discharges, radiation; into  complex structures.  These are part of a highly specialised chemistry called the chemistry of evolution .One mechanism for increasing the concentration of the organic reagents of the primitive soup predicts the absorption of these composites by inorganic particles and clays. In particular the absorption by minerals would have also had the task of impeding or diminishing photolythic and pyrolythic action favouring stereospecific catalysm. A proof of all this is the synthesis of the polypeptides of the glycocolla starting from aminoacetonitrile absorbed by kaolin. Melanin must have played and important role in preenzymatic chemistry. This black substance continues to be synthesised by the living organisms of the Earth today, and is present in every Phyla. It is possible that, hundreds of millions of years ago, there were two solid phases on Earth, one inorganic and one organic. The melanin is thought to be the organic solid state formed by a radical process catalysed by light and by temperature. This process is still observable in our environment which is subject to radiation and continuous electrical charges. Melanin must be considered not only as a "lattice" but also as a semiconductor. In our opinion in the presence of an atmosphere which was both reducing and oxidising, with electrical discharges, solar radiation at short wavelengths and sudden thermal changes atomic collision, melanin was the birthplace of aromaticity. Melanin, through its large surface, its porosity and its internal molecular sites offers a complex but satisfactory situation for stereo-specific or even metalorganic synthesis. The radical property of melanin is important for the chemical evolution on Earth. Uniformally distributed unpaired electrons in melanin contribute to the creation of organic-metal semiconductors which interfere in organic synthesis. It is important that water can reach the unpaired electrons and condition the relaxation time. In the chemistry of evolution it is always a risk to say which, among all the molecules, is the first, the most primitive.  Up to now it seems clear that the role of the semiconductors in a biological context must also to be considered. Although light has been thrown on the subject recently, there are still several doubts on the biogenesis, the structure and the function of melanins. We hope that this work is not only a testimony, but can contribute to improving our knowledge. While the study of crystalline substances has achieved important results for molecular structure the organic chemist finds himself at a disadvantage in front of dark, amorphous, insoluble, non crystallizable substances which are fluctuating and non-reproducible.

Melanins in microrganisms.

( Allomelanins ) ( Link 15 )

Bacteric melanin (a limited number of species and genus Bacillus, Pseudomonas, Azotobacter) are seen as black, dark red or yellow substances with different tones and rarely are even green. They are insoluble and amorhphous with non-specific spectral characteristics, but, as always, with a constant EPR signal. The micro-organisms which produce melanin belong to aerobic forms . The formation of melanins in micro-organisms is an oxidative process of phenolic substances, influenced by radiation, temperature and the presence of metals. In the future the cellular and extracellular melanins of the micro-organisms could represent a very interesting material to study for molecular structures and organic synthesis.

Besides DOPA and cysteinyldopa ( Link 14 ) , other polyphenols including non-nitrogenated forms are responsible for the formation of black pigments in micro-organisms. Typical melanogens of microrganisms are, for example, pyrocatechin, 1-8 dioxynapthalene and (+)-scitalone isolated in the culture of the brm-1-mutant of Verticillium Dahliae. A characteristic of some micro-organisms is that the formation of the pigments appears as a disintoxication reaction of the phenols present in the old culture, perhaps because of a mutated metabolism inside of the cell. Micro-organisms (Azotobacter chroococcum) which contributes to the formation of the humic acids and the humus of the ground and participate in the general life of living nature. Ample and in depth studies on Aspergilline, the black pigment of Aspergillis niger, have been made starting from Quilico  ( Milan ) and continued by his school: The tyrosinase enzyme was isolated from fungi for the first time and today this is still considered the main enzyme in the melanogenesis of all the living species. Tyrosinase has been given this name because it is able to catalyse the transformation of tyrosine into DOPA. For many fungi though, for example in Neurospora crassa, the enzyme tyrosinase does not belong to the category of the enzymes necessary for development. The synthesis of tyrosinase can be induced in such fungi, as for example in Glomerella cingulata. Among the biological functions of the melanin there are those of defence of the cells from different types of radiation (which acts as a rapid ecological selection factor), and defensive lysis for survival in pigmented fungi surrounded by a microflora in biologically active terrain. The melanogenesis of the tuberculosis bacillus is unclear as is that of the Mycobacerium nigrum. The Mycobacterium leprae does not produce melanin in situ but provokes a pigmentation of the skin of lepers. The melanin isolated by the Bacillus salmonicida is of a red-brown "colour". The melanin produced by micro-organisms may be either extracellular or intraccellular. Intracellular melanin is the most common and is firmly linked to the cell walls. The combination of the environment and, as always occurs in radical processes, light and temperature, influences the production of the pigment. An antibiotic activity has been attributed to some of these pigments (blacks, browns). The brown melanins produced by these organisms are similar to the humic acids for a series of chemical and physical properties. Many microscopic fungi with brown and dark structures (hyphe, spore, conidio, peritecio etc.) are known even if they have not always been shown to be melanin. Among the fungi producers of melanin are believed to be: Pullularia plullulans, Cladosporium mansonii, Phialanphora joanselmii, Nadsoniella nigra var. besuelica, Neurospora crassa, Aspergillus nidulans, Daldinia concentrica, Aspargillus niger, Cephalosporium sp, Mollisia caesia, Ustilago maydis DC, etc. Tyrosinase is very common in micro-organisms, but its presence does not always mean that the fungus produces melanin. It seems that it is also possible to produce melanin in an anaerobic environment.

Melanins from plants.

The formation of brown, red-brown and black products is a phenomenon which is often observed on petals and leaves. The melanogens of plants are often non-nitrogenated substances . The non-nitrogenated melanins of plants are called allomelanins. There are also pigments which form because of damage to the tissue (potatoes, apples, banana etc.). It is known that on contact with atmospheric O2 cellular phenols and polyphenols produce complex dark and brown products, in a non-enzymatic reaction

Different Compositae and fungi contain acetylenic compounds which may go black under the action of light . To date, the melanins of plants have not been given serious chemico-physical examination. A plant rich in melanogenes is the broom Sarothamnus scoparius. From legumes and from fruit it is possible to isolate tyrosine, DOPA, tyramine, DOPAamine, epinine (N-methyldopamine). The blackening which is seen in the course of maturation and conservation of bananas seems to be due to the neurologic melanogene DOPAamine. The typical blackening of slices of tubers (potatoes) and fruit (apples, pears, etc.) and plants (in the Vicia faba one finds large quantities of DOPA) seems to be a process of melanogenesis from tyrosinase or DOPA. In all the plants the polyphenoloxidases are very common but this does not always mean the formation of melanin. Besides, while the melanin is localised, the enzymes are much more dispersed in the various parts of the plant.

 

Melanin in Animal kingdom.

 

In the animal world melanin is always present in various forms and coloured from black to vermillion and to yellow. There are innumerable shades of colour: brown, red-brown and yellow which depend on various factors . The black animal melanins have been given the name eumelanins and those of lighter colours pheomelanins (Link 14)  . The various shades of colour which we observe in nature depend on the gap ( Lin9 ) but also on the purity, on the state of oxidation of the aggregations, on polymerisation and on copolymerisation of the various melanogenes or other substances. Some species of pigmented animals contain black granular pigments in the endoderm while a brown pigment is quite common in the ectoderm. Particular interest has been seen in the case of melanogenesis of the cuticola of the insects (sclerotisation process). In some studies, carried out in the past and neglected in the present, on the cuticola of the desert locust, Schistocerca gregaria, well known for the economic damage which it provokes in poor populations, it was shown that the melanogeneses and sclerotisation are two distinct processes. The melanins of vertebrates and invertebrates can be observed in the skin, in fur, in hair, in feathers, in scales, in the choroid, in the peritoneum, in the pia mater, in the brain, and in melanomas (malign tumours of an intense black colour). One cannot yet confirm that the various melanins coming from different sources are the same, even though they have the presence of a free radical and the potential capacity to conduct electrical current in common. Sometimes animal melanin has the function of protecting the skin from radiation (short wavelengths), of controlling temperature, of mimetisation. Little or nothing is known of the structure and the functions of neuromelanins (eg. substantia nigra) expecially with respect to the elecrical properties of this pigment (Link 22). Obviously the melanin associated with tumours has been studied more, by biologists.  

Cells of Substantia nigra from normal individual stained with toluidine blue.

Normal cells of the Substantia nigra stained with toluidine blue

The melanins of mammals are deposited under the form of granules in specialised cells called melanocytes, rich in iron, copper and zinc. The granules have an ovoid form (0.1 x 0.4 m - 0.18 x 0.6 m) or spherical form (0.2 x 2.2 m), of proteins covered by melanin. The protein can be "discovered" with a bland attack using H2O2 which "dissolves" the melanin (1, c). It is not easy to recognise a melanin and it is almost impossible to do so when the melanins are formed by copolymerisation from different precursors. The way in which the melanin is seen in the animals is fascinating, with geometric figures of rare beauty. The way in which they produce blue and green colourations is another fascinating argument (Link 9, 11). One need only think of the alternation of colouration, the lively tints, the shades, the tones which, with the exception of man, make the animals so pleasant to see. The mutable colours which many animals use to hide from predators are surprising; in these colourations the conductor melanin has always played an important role. The melanins are responsinle for the different "structural" colours like the Tyndall blue which is seen in the skin of fish, amphibians, reptiles and birds. Through differing mechanisms and the Tyndall effect melanin produces the blue, which in the presence of a yellow pigment (for example a carotenoid) gives a beautiful green colour. In English schools the teacher tells the students to treat a green feather from a bird (e.g. a parrot) with CS2, leave it over night, remove the intensely yellow coloured CS2, wash it with H2O, dry it, and you have a beautiful blue colour.

Marsupials arrived in Australia from South America via Antartica. Almost all the species have a red-orange fur (Megaleia rufa) or grey fur (Trichosurus vulpecula) ( Link 14 ). In these animals there should be a double melanogenesis, one from cysteinyldopa and one from tryptophane via kynurenine, with the formation of cinnabaric acid. A double melanogenesis is also present in the cephalopods and in some insects. A substance with a structure similar to cinnabaric acid has been isolated from the eyes and from the skin of the cephalopods and from the eyes of Musca domestica. In the extraction mixture this substance transforms into various composites, among which dihydroxantomatine has been idientified which could be a key intermediate of melanogenesis and pheomelanogenesis from kynurenine .The characteristic melanins of the hydrosphere and the lithosphere belong to humic substances. They form from various precursors, like phenols, sugars, aminoacids, glycerides, through the combined effects of radiation, heat, and micro-organisms. Considering the variability and complexity of their origins, the structure of the humic acids is one of the most complex problems in chemistry. For the humic acids present in the lithosphere and for those in the hydrosphere composition and structure depend on the place where they are found and how they form. The humic acids play a role in the biogeochemical cycle of carbon, though it is not clear how . Melanins are normally present in organisms which live in the acquatic environment: both in animals and in vegetation. Often melanins are the dark background which highlight the silvery colour of many fish.

Pyrrole-black.

BSM

Even though it has not yet been recognised as a biopolymer pyrrole black is of great interest for structural studies and for the chemico-physical behaviour of natural melanins. Pyrrol black can be prepared by chemical, electrolytic or ultrasound oxidation. If one examines the polymerisation process of pyrrole in peracetic solution (Angeli) following the evolution of the solution over time, the EPR signal can be observed from the beginning and becomes more evident at lower temperatures. After a certain fluctuating and non-reproducible time one obtains a black precipitate characterised by an intense EPR signal but wihout any hyperfine interaction. The presence of unpaired electrons is necessary for the formation of conduction bands and for the presence of the fundamental colours of the semiconductors, black, vermillion and yellow. Another necessary situation is that the radicalic species are stable and permanent, as happens in the natural melanins. Important structural information for the history of natural black systems is the fact that electrical conductivity in an organic material is linked to the existence of a polyenic or a congugated polyarenic system . It has been demonstrated that the polypyrrol contains cation centres; it is a polycation with spectator counteranions. The conductivity of these systems depends on the method of preparation, on the type of counteranion and on the extension of the planarity. In the preparation of pyrrol black the counteranion, also called the spectator ion, is also taken from the medium of the process. The black produced varies in conductibility, and in the number of cation centres present . The anions ClO4-, BF4-, AsF6- and HSO4- give good conductors. Acetylene black is also a polymer semiconductor and presents cation centres with spectator counteranions . Since there is no reason to believe that natural melanin does not belong to the category of the polyarenes and polycationic polyenes, like pyrrol black and acetylene black, it is necessary to review all the chemical and biological analytic data gathered to date in the study of natural melanins (eumelanins, pheomelanins, allomelanins). Besides it is highly probable that the natural melanins are artefacts with counteranions provided by the medium in which the "purification" has been made. If one considers the black from 5,6-dioxyindole one finds interesting data. 5,6-dioxyindole is among the most important melanogens and is a continuous reference for the chemistry, biochemistry, and biophysics of the eumelanins. The oxidation product , generally a black or vermillion precipitate, is isolated through acidification with H2SO4. The analytic results which are obtained worry authors, in that they do not agree with the theory which predicts that the blacks or vermillions are polymers of the 5,6-dioxyindolequinone of its derivatives. They claim that a certain number of molecules of H2O are linked to the polymer, in an "ad libitum" or random quantity and in such a way as to close the gap between the analytic data and the theoretical calculations for a polyindolquinonic ‘’ polymer’’.

 Several other blacks like the analin- black, catechol- black, 3-aminopyrocatechine- black and tryptophane- black are in the same class as these composites. Polyphenols composites, as is known, play and important role in the melanogenesis of humic acids.

Physics and Chemistry of Melanins.  

The peculiar characteristics of the melanins are always the colour and their free radical nature (6). Scarse results have also been found with the most sophisticated apparatus (X-rays, MS, NMR, Laser) (Link 22).

There are three fundamental theories on the chromophores of melanins. The first is that which proposes that melanin is a mixture of chromophores which resonate in different parts of the spectrum. A mixture of red, blue, green etc. chromophores absorb the radiation of all the wave lengths of visible light and appear black. Another theory predicts that a highly conjugated system produces black, brown and red-brown depending to the gap value of the semiconductors. What is the extension of the conjugation that allows a body to appear black? Many natural substances possess extended systems of conjugated orbitals (for example the carotinoids) but none of these compounds appears black. The study of the electronic structure of an ordered and ideal polymer of 5,6-indolquinone which is a common component of the eumelanins (hair, eyes, skin, etc.), conducted using the theory of Hückel, predicts that the black colour appears in a polymeric unit with a number of monomers higher than 10 and up to a infinite polymer and that, besides, it is an amorphous semiconductor and a good conductor of electricity (3) . The theoretical model is not supported by few experimental data. The only experimental data which currently exist for polyarene and polyene structures are those relative to pyrrole- black and acetylene -black which, as has been said, are radical-polarone system. ( Link 9, 12, 21, 22 ) In the study of the melanins it has often been claimed that the different colours, yellow, orange, red, red-brown, black are due to the type and the size of the granules of the pigment and to the distribution of the granules in the tissue. A third theory proposes that the melanins are schemochromes, that is that the colour depends on particular   particle structure. The melanins could be a sort of black body in which the light which penetrates is reflected and diffused until it is completely absorbed. The cause which produces the "black" must be studied using solid state theory and band model of semiconductors.

Melanin as stable free radical .

Despite the fact that every chemical and physical study of melanin must be interpreted with more care compared to what would usually be the case in the study of pure and crystaline substances, the black melanins (eumelanins), brown, red-brown, and yellow melanins (pheomelanin), the allomelanins and the humic acids present a characteristic EPR signal, sometimes with some hint of a hyperfine structure  (Link 22). The origin of the paramagnetism is still a controversial problem. Several efforts, (nothing is ever clear and definitive in scientific research on the black substances), have been made to correlate the free radical nature of the melanin with certain biological functions, like the physiology of vision, photoprotection, threshold switching, the electret effect etc. Studies have been carried out on all the melanins in acqueous suspensions and almost always give an EPR signal at about 4-6 G. The spin concentration is around the value 4-10 x 10¹⁷ spin/g. In the "polymer" there would be one free radical every 200-1,000 "monomers". It would seem that there are two radicalic centres in the black products that originate from the o.phenols: one being essential (intrinsic), highly stable, generated in the course of melanogenesis and "trapped" in the product and the another being extrinsic, transient and reactive which can form in the melanin by the action of the different chemico-physical agents. Passing from black melanins to brown and red-brown products (pheomelanins) it is possible to observe radicals with better defined structures, at different pH, like those of semiquinonamine and semiquinone. EPR studies carried out on the hair and skin of several bovine races and on albinoes have mainly been used by geneticists and pathologists. Albino subjects, with the same phenotype character, have hair with differing electronic characteristics. In some albino subjects there is a weak EPR signal which is completely absent in others. There are, that is, true albinoes and false albinoes (6, l).

The chemistry of free radicals, which has almost always been associated to the processes of polymerisation and oxidation, has developed in isolation from the context of organic chemistry. The chemistry and biochemistry of natural substances have always considered the free radicals with diffidence because of their reactivity which makes them difficult to control. It is probable that different radicalic reactions intervene in melanogenesis. Melanogenesis has never been considered as an essentiallly radicalic process. The EPR signal, present in all the melanins, has been attributed to a system like the cyaninic colourants (merocyanines) which give, as is known, EPR signals similar to those of the melanins even though they do not have unpaired electrons. This explanation of the meaning of the EPR signal by the merocyanines seems strange? The hypothesis that the signal comes from an inert radical of the copolymerisation material of the melanin cannot be accepted. A somewhat surprising observation is that the radical is little present in the melaninin granule and therefore of little significance , while it is highly probable that the important properties of the melanin (solubility, colour, reactivity, conductivity) are linked to this electron which lives alone in a gap of the granule. When the melanin lightens from black it becomes brown, red-brown, yellow, that is one passes to pheomelaninic granules (hair, fur, feathers, eyes), not only is the EPR signal always present but it appears in a more and more structured form, to the point that it is possible to distinguish the black (melanin) from the red-brown (pheomelanin). The EPR signal is present both in melanins prepared in the laboratory under most varied conditions and in pheomelanins of very varied origins. For the humic acids  of the hydrosphere and of the lithosphere, so important for life, one has a very similar general picture even if the heterogeneousness of the material makes the granules of the humic acid much more difficult to study. The results obtained with IR, NMR, X-rays and MS cannot be believed significant. The various spectra which often present absorption characteristics, may not be due to the humic acid but to incorporated substances (which can be used for the qualitative and quantitative analysis of terrains and of waters). The EPR signal is pure, clear and very similar to that of the melanins. In general one obtains the EPR signal of two types of stable radical which must be attributed either to the pyrocatechine-resorcine type or the quinhydrone type. In general one has signals of various intensity according to the origin of the humic acid (humic acids from micro-organisms, soluble humic acids, black carbon humic acids, grey humic acids, brown humic acids, marshland, manure, peat). Notable paramagnetism is present in the fungal humic acid (Cephalosporium gordoni) which is also darker: the blacker the composite the more intense the EPR signal. It is calculated that for a concentration of 10¹⁷ spin/g in a "composite" of molecular weight 10,000 there is 1 radical for every 600 monomers. It is plausible that, from a structuralis chemical point of view, the part of the "macromolecule", perhaps the chromatic part (acetylene-black), responsible for the paramagnetic phenomenon is assimilable to that of the more typical melanins present in the animals or produced by micro-organisms. Therefore the history of the EPR signal is identical for all the melanins regardless of their origins. As often happens in research the failure of an objective (identification, for example, of the structure responsible for paramagnetism in the melanin) produces new and useful knowledge. The discovery that the humic acids of various origins and natures are free radicals has given useful information on all the existing relationships between the EPR signal and the properties of these substances. In the soil there are all the conditions for the genesis of free radicals: water, light, heat, organic and inorganic catalysts (Fenton reactive types). The soil is all a melting pot of radicals; in the soil the primordeal matrix can operate with ease .

The hypothetical radical melanogenes of the soil can form through the action of oxygen and light on products deriving from micro-organisms, from the oxidative demolition of the lignin, and from other biological polymers. In the physiology of plants humic acids act both as growth factors and as activators of cellular respiration. The semiquinonic radicals of the humic acids influence germination and, linking to atmospheric oxygen, transport active oxygen in the soil. The humic acids as polycations fix and transfer precious counteranions to the life of the soil and the waters.They can be good conductors when opportunely prepared and doped. In conclusion, polymers like melanin from DOPA, melanin from sepia, humic acids, melanin from phenols, like pyrocatechine and pyrrole blacks, etc. , apparently differing among themselves, exhibit an identical signal at EPR which can be reinforced up to even 100 times if the measurement is made on sodium salts. The melanins of the soil (humic acids) form by a fluctuating and non-reproducible mechanism where time must be considered as an intrinsic parameter of the dynamic of the natural process and where the activation of water is a fundamental parameter (also cellular liquid). The concept of time is a problem of fundamental importance for the biologist of melanin, in that in its genesis this is not free from environmental and cosmic forces and influences or from the activation of the water under the action of electromagnetic fields or from solar phenomena in general. To show that some reactions cannot be located in the current logic of science, Piccardi  stressed that accepting time as a coordinate negates the fundamental dogma according to which only reproducible experiments are valid. In the case of melanin, in fact, it is not possible to control the conditions in which an experiment is conducted without taking into account the time coordinate, because during its course the conditions in which an experiment, a chemical or biochemical reaction, is conducted change. In experiments on melanin the time may not be considered an isotrope in every direction in space, nor homogeneous for every successive instant. Melanin cannot be represented by a formula, with a complete melanogenetic scheme in that it is not representable in terms of linear equations because it is not possible to hypothesise a correspondence between cause and effect in the temporal succession of the relationships. A negligible reaction in a fixed instant can become a determining reaction in a succeeding instant.

In melanogenesis many classical concepts, definitions, chemical dogmas are obsolete, they are lost in the past. If the experimentor looks at the scientific explanation of the natural processes and is not content with the measurements of the immediately measurable quantities then he understands that he is working in an imperfect way. The analysis (combustion) of the melanins coming from the sack of the sepia are non-reproducible even with the same method of preparation of samples coming from the same source. To the chemist the synthesis of black content in the sack of the sepia must seem a non-reproducible process and is perhaps fluctuating because of the interference of magnetic activity and radiation of the sun on the sea, of the atmospheric electrical potential, of the variations in the Earth’s magnetic field.

The spirit of research in the field of the melanins must be renewed, accepting the hypotheses works some researchers, sometimes students, most of the time young emarginated researchers, would like to introduce into the world of research.

MELANOGENESIS

The chemical processes which produce black substances in vitro, either in the presence or the absence of enzymes are considered as chemical melanogeneses. Three parameters dominate the scene of mealanogenesis: colour, conductibility, EPR. Michaelis was the author of one of the most important and general theories on two-step oxidations in organic chemistry (1932). Oxidations and reductions of organic composites (in vivo and in vitro) are bivalent (bivalent reactions) and these reactions procede necessarily through two successive univalent steps. A radical process must be at the basis of the synthesis of the melanins (6, n). In the case of a quinoid substratum Michaelis, applied the term "semiquinone" to an intermediate electronic form called "free radical " . The existence of these electronic forms was proved through the accurate study of free radicals which are formed during oxidation. After the death of Michaelis a not indifferent advance was made in the semiquinonic free radicals which became better identified and classified by EPR studies. In the study on oxidation of hydroquinone it was shown that the formation of the semiquinones is a reversible reaction at neutral pH, while a black precipitate is formed at alkali pH. The black precipitate which is thereby obtained has an EPR signal similar to that of the natural melanins. The black precipitate which is obtained, for example, by the oxidation of DOPA is the result of the polymerisarion between quinones and phenols. A question which can certainly be asked is: How come the radicalic mechanism which Michaelis has predicted in oxidation does not occur in the case of the melanins? However, everyone who has studied the formation of the melanins with EPR, starting from the diphenols like pyrocatechin, has registered EPR signals which transform in the course of melanogenesis over time. This suggests that different chemical species, also coming from oxidative and bioxidative degradation, are produced in the formation of melanin. An observation which successive studies have made in the fundamental equation of DOPA-melanin, which is considered the fundamental melanogenesis of superior organisms, is that in effect there is more oxygen in the black than that which the theory can predict and that besides the composition is variable and fluctuating. These results are the same whether the oxidation of the DOPA is made with or without enzymes.

It should also be noted that often the experiment has been carried out with the use of tyrosinase from pathological sources like melanomas.

Oxidising the DOPA with or without enzymes forms different intermediaries like dopaquinone and dopachrome, 5,6-dioxyindole (DHI), 5,6-dioxyindole-2-carbonic acid (DHICA)  which can be artifacts.

If the oxidation is followed by UV one sees the formation of a species which absorbs at 280 nm to which the structure of the dopaquinone has been attributed, and which, with a kinetic of the first order, transforms into a species absorbing at 480nm normally identified with the dopachrome. This reaction is strongly dependent on the pH. Oxidising the 5,6-dioxyindole even with simple atmospheric oxygen forms a red substance in solution (540 nm) to which the formula of 5,6-indolequinone or one of its dimers or trimers has been attributed. Anyway the reaction is rapid and leads to a black even in the absence of the enzyme. The first stages of melanogenesis seem typical radicalic reactions deriving from a semiquinonic radical of the DOPA. The formation of phenoxonic radicals is well known in living organisms and explains the formation of metabolites of plants like the alkaloids, the lignin etc.

Besides, in the course of pheomelanogenesis  a series of cysteinyldopas ,6-cysteinyldopa (yield 2%), 5-cysteinyldopa (yield 70%), 2-cysteinyldopa (yield 15%), 2,5-dicysteinyldopa (yield 6%) form by reaction between DOPA and cystein. The genesis of these substances is well explained with the possible reactivity which the mesomeric radicals of the DOPA. The dopasemiquinone which is formed in the first stages of melanogenesis by oxidation of the DOPA also provides the explanation of the formation of different tricochromes (pheochromes) (1, d) from the various cysteinyldopas. Recently it has been shown that the oxidation of the 3-oxy-kynurenine also procedes through the formation of free radicals . An examination or a reexamination, with EPR, or radical sequestrators which lead to establishing the participation of the free radicals in the course of melanogenesis both in an autooxidative process and in an enzymatic process would be welcome; in this future research it is necessaty that the enzyme is compared only with its natural substrate. Melanin forms by a radical process, but surprisingly it can degrade with a radical process, by the action of light, of atmospheric oxygen and of the H2O2 which is formed. Like in soil the radicals play a very important role for the biology of the skin. The light , the environmental factors, the fluctuating factors of the cellular water are very important. The periodic influence of the nuclear explosions of the sun make the melanogenetic system of the skin one of the most interesting biological phenomena not studied on the Earth.

 In conclusion a short comment on melanogenesis in mammals. It seems that recently our knowledge in the field of physiological and pathological melanogenesis in mammals has progressed . Unfortunately no distinction has been made between the melanogenesis under strict enzymatic control, as claimed by the authors  of melanoma and of neuromelanin isolated from every environmental conditioning, and the radical fluctuating and non-reproducible melanogenesis of the skin. The formation of black products in the cells is illustrated by one scheme denominated melanogenesis which is based mainly on work started in 1924 by the physiologist Henry Stanley Raper of Manchester . The chemical intuitions of the researcher not common for a physiologist were certainly influenced by Sir Robert Robinson then professor at Manchester. The history of melanin can also be seen in the history of schemes of melanogenesis but that would take us too far afield.

Black conductors

In 1964, based on BCS calculus (Barden, Cooper, Schrieffer theory of superconductivity) W.A. Little proposed a chemical structure for a consistent ideal organic superconductor, composed by a polyunsaturated chain, called “spine”, substituted in some points by heterocyclic structures (often resonance hybrids) having cation centres and counteranion. Even if the hope finding a room temperature superconductor has not been realized, Little’s hypothesis had been very profitable, making possible the synthesis of numerous compounds or organic materials which proved to be conductors. Because of their technical properties these compounds were given the exotic name “organic metals” You can therefore confirm, from experimental data that polyunsaturated polymers (the “spines”) with cation and counteranion centres (and vice versa) are conductors or candidates for conduction. Many of these materials, for example pyrrole black , were considered for long time as insulators but experience has shown that the conductivity of Little compounds is influenced by various factors such as the way the polymer film forms, the doping, type and concentration of the counteranions, the pH, temperature of reaction, etc. The significance of the colour of these products or materials and their possible correlation with EPR and electroactivity have never, to our knowledge, been looked into by organic chemistry. The present paper attempts to find this correlation and to create a bridge between physics, chemistry and biology. In this connection, it should be stressed that conductivity also plays an important role in various biological mechanisms and sites, where these polymers are present. It is well known that a pure semiconductor  can occur in coloured forms originating from the transition of electrons into materials with band structures, whereas most synthetic and natural colours are attributed to the transition from one molecular orbit to another.

A pure semiconductor may appear black red yellow depending on the amplitude of the prohibited band: the colours of the band have generally been attributed to inorganic compound with different. eV expressed values (E.g. CdS 2.6 eV yellow, HgS 2.1 eV vermilion, CdSe 1.6 eV black). The sequence of the colours of the prohibited band in increasing order of this amplitude is black-red­yellow-colourless, the rarer green. Some of these pigments, with different chromatic shades, occur in nature and are called biochromes . Furthermore formation of a light-induced charge-transfer complex was observed by measuring the increase in the conductivity after mixing solutions of chlorpromazine and sepiomelanin [23 page 22].

A polyarenic structure with cation and counteranion centres, is a structure containing all the structural elements necessary to obtain a Little conductor or a polymer candidate for superconduction. The polyarenic spine present in sepio­melanin and in other natural compounds is that of acetylene black . We found that numerous families o[ polymer conductors, some of biological interest, descend from three well-known systems, namely, indole, pyrrole (desbutenylindole), and benzene (desaminovinylindole).

The chemical formulas of the precursors of the product or the electroactive material are reported, together with the polymer colour as it appears in nature or in the laboratory. Moreover, one can find the relationship between these compounds (materials) and natural pigments, the observed electroactivity, (with the term “positive” and the theoretical electroactivity with the term “candidate”), and the presence of an EPR signal common to all the pigments. The polymer material with electrical activity indicates an intrinsic or extrinsic conduction (doped or undoped).

In the most polymerized phase the derivatives of pirrole , indole , benzene , are amorphous black products. The derivatives of indole are predicted from calculus to be amorphous semiconductors . The derivatives of pyrrole, like pyrroleblack , bile pigments  and porphyrins  are conductors. Among the derivatives of benzene there are various good conductors, with electrical properties whith can be improved they are doped  such as aniline-black [, poly(p.phenylene), fullereneblack  and graphite. Fullerenes and graphite, which do not seem to be Little’s structures, are been regarded as arbitrary polymers of benzene based upon their colour, EPR, electrical activity. Both calculus and Little’s theorem indicate that all the melanins (derivatives of pyrrole, indole and benzene polymers), BCM or BSM are amorphous semiconductors. With increasing the number of rings, the aromatic character decreases, whereas the radicalic behaviour, the reactivity towards oxydants and other chemicals, the electron paramagnetism(EPR) and the electric conductivity increase. At the same time there is a colour shift from colourless (benzene) to green-black (heptacene). The EPR signal can not correlate to a specific structure. The properties of the polynuclear compounds gradually approach those of graphite, with the fullerenes being found in the middle of the series. This also applies to electrical conductivity. Aromatic polynuclear hydrocarbons are semiconductors and exhibit photoconductivity, a strong increase of conductivity also being recorded under the influence of irradiation. Consequently, a photocurrent is produced, which is caused by a conduction band, as in graphite.

  The close structural relationship between the aromatic polynuclear fullerenes and graphite is well expressed by the colour, the EPR signal, the electrical conductivity, and the sensitivity to oxygen. As a matter of fact graphite can be considered the most highly condensed aromatic system, or the prototype of aromatic compounds  and this justifies its position  as a “polymer” of benzene. From an electrical point of view, the differences between conducting polymers and graphite like compounds are to be ascertained. The relation between colour, molecular weight, and conducibility is not known. A new class of carbon compounds obtainable from laser vaporization of graphite can be listed: fullerenes [13] (from C₆₀ to C₁₅₈ so far obtained) are good conductors in a doped state. C₆₀ has a beautiful magenta colour whereas C₅₈₀ is, from a theoretical point of view, expected to be black.

  It is of interest to note that: «it was shown that some of the black dust clouds of the Milky Way Galaxy were full of long carbon chain molecules (the polyynes). Not only was HC₅N detected but subsequently, the even longer chains HC₇N and HC₉N were discovered. The discovery of fullerenes  was an important breakthrough in our knowlege of the carbon content of space. The fullerenes come from the Giant Red Stars which produce large quantities of “carbon molecules” and carbon dust. The prototype C₆₀, the most studied of the fullerenes is a polyhedral cage with pentagonal and hexagonal faces.  The well studied poly (p.phenylene)  which shows different colours, shades of black, good conducibility in a doped state presenting all the structural features of Little’s model. Pyrroleblack is a good semiconductor in the doped state [2, 4], for which a linear and planar chain structure, linked through the 2,5-positions of the heterocyclic ring, is generally accepted. This structure was confirmed by the isolation of pyrrole acids (during oxydation with KMn O₄ )like other blacks, pyrrole black gives a strong EPR signal and contains cationic centres with counteranions. Is after melanin, according to Pullman , is one other similar case of a “bonding empty orbital” the biliverdin, a metabolic degradation product of hemoglobin, Bile pigments  must be considered, for their structure and properties, candidates for conductivity and superconductivity. The inverse, namely the transformation of a bile pigment into a porphyrin, was accomplished treating the bis-iminoether of bilirubin diethylester with cobalt or nickel salts, yielding the cyclic tetradehydrocorrin .

 In the purple ink of the sea-hare Aplysia the biline  derivative aplysioviolin is present. The uroporphynin I was found in the integument , whereas the giant neurons of Aplysia are coloured by biline derivatives. Some of those pigments are expected to be conductors or candidates for conduction.

It would be obvious at this point to think of fish producing electrical current (torpedo, electrophoros, astroscopus, malapterus etc.): but no knowledge of conducting polymers is found in the literature . The melanins are often considered macromolecules made up by the cross­linked skeleton of the monomeric melanogens with a variable number of non-localized free electrons and positive charges, diluted along the surface of the polymer and balanced by counteranion spectators. This structure has some structural and functional analogies with acetylene-black , which can be considered a simple unsubstituted melanin. All indole polymers and derivatives are Little’s structures and therefore must be considered as conductors or candidates for conductivity.

Neuromelanin has been recorded to be originated from dopamine and cysteinyldopamine, through a mixed enzymatic and non enzymatic oxidative process, catalyzed by Fe and other redox metal ions present in substantia nigra. Neuromelanin appears to be associated with the bioelectrical activity of neurons, with the degeneration of the substantia nigra and Parkinson’s disease.

Studies conducted on the melanins have, to date, not given satisfactory results (Link 21, Link 22). This state has been reached for two main reasons: the first is that Biology, Chemistry and Physics have developed in unidirectional and unidisciplinary ways; the second is the ignorance of the fact that almost all natural black pigments are amorphous semiconductors which in nature are formed by fluctuating radical processes  and not by enzymatic processes. 

In a previous work on biochromes we hypothesised, on the basis of experimental data and theories expressed in the literature, that many natural pigments owe their colour to electronic transitions in band materials (www.organicsemiconductors.com). Our working hypothesis is that all black pigments, both synthetic and natural (melanin), belong to the organic solid state. The pigments are characterised by the colours black, blue black, matt black, pitch black, brown black, copper black, bronze black, gold black, metallic black being those of the pure semiconductor. While the colours of the pure semiconductors are well defined in terms of the amplitude of the gap the situation is not as clear for the amorphous semiconductors where the “colours” black, brown, brown-red predominate. The colours of the amorphous semiconductors are often seen in hair and body hair of various mammals . These pigments although not pure have characteristic properties of the amorphous semiconductors:

a)    weak electrical activity(10^-11 – 10^-7 W^-1cm^-1) at the limit of the insulating state. The conductivity is increased by doping, by the type of counteranion, by the formation of charge transfer complexes, methods of synthesis and purification used, by light and temperature.

b)    A stable EPR signal

c)    The phenomenon of threshold switching

d)    The photovoltaic effect

 

.

How can the electroactive material be ‘a priori’ synthesised? In order to plan the synthesis, especially concerning the superconductor material at high TC (critical temperature of transition from semiconductor to superconductor) the following characteristics must be considered:

a.    stable free radical

b.    obtain band overlaps; small HOMO-LUMO gap, large band widths

c.    delocalized molecular orbitals

d.    not homogeneous charge and spin distribution.

e.    segregated stacks or sheets of radical species.

f.     no periodic distortion which yields a gas in the density of states across the entire Fermi surface

g.    little disorder

h.    molecular components of appropriate size

i.     fractional charge transfer

1.    strong interchain coupling

m.   polarization species to help to reduce U.

The conductivity of melanin in its natural state is rather poor but what can influence the electrical conductivity may be the presence of foreign substances or metals (doping effect )  or a proteic part which is difficult to dislodge from the complex. It is also to remember that heating modifies planarity of the system (hydrogen bonds and conductivity). In melanins iron is always found as well as other metals such as Zn and Cu; the latter is part of the tyrosinase but it is found in the premelanosome and in the melanosome in much smaller quantitles than Fe. The presence of iron suggests that the radical process is triggered by a peroxidase/H₂0₂ system or by a Fenton reactive system. The process of melanogenesis is a fluctuating process which transforms a colourless insulating compound into a black conducting material or into an amorphous semiconducting material. In the course of the process there is a dramatic moment of transformation of the orbital transition colour into colours of the electronic transitions of band materials, the transformation of the molecular state into the solid state, the transformation of the molecular biology into the biology of the solid state. Theoretically based on the existing relationship between conductivity and the number of double bonds, on the limiting number of indole units seen with MALDI-TOF-MS the transformation could occur when there are about 30—40 linked double bonds of the polymer present. These compounds for polymerization yield black products which are electronic conductors, especially when doped. This was also the first attempt to represent the organlc amorphous solid state. The electronic structure of the amorphous solids can be written in terms of bands of energy and prohibited intervals as for the crystals, microcristalline structures, and highly repetitive and stereospecific polymers. The electronic, magnetic, optical, structural properties which interest the organic state are not molecular properties but are, rather, associated with intermolecular interactions. Blacks such as graphite, aspergillin and Daldinia-melanin belong, as shall be shown later, to polycondensed benzene systems which when doped, are electrical conductors. The fullerenes (or melanins from space) also belong to polycondensed systems. There is a relationship between colour, the number of double bonds (length of the Little spine) and electrical conductivity  Our knowledye of  the organic polymers were, until a short time ago, considered to be insulators. The relation between the structure of a polymer and its ability to transport electrical charge is still today limited by technological interests, and therefore is based on data which are not altogether satisfactory for the study of structure. In a previous work (Nicolaus, B.J.R., et al., 1997) we tried to find a relationship between the structure of a natural organic compound and its conductivity. The use of the theory of orbitals is often mixed up with band theory without major errors. In a polyenic chain the n-electrons are delocalized and this tends to make all the -C-C- links of equal length. This is seen in the polymers of type:

                                              

 

where the conductivity increases wlth the increase in the number of double bonds. Melanin-like polymers with conjugated double bonds (melanins) are, in general, poor conductors unless appropriately doped.  Melanin-like polymers with conjugated double bonds are, in general, poor conductors unless appropriately doped.  The crystalline ®  amorphous transformation is observable with UV, IR, EPR spectra which, with the increase in polymer length, tend to become broad spectra. The conductivity is seen to vary between the crystalline oligomers to the amorphous oligomers. The electronic structure of the amorphous semiconductors is still describable in terms of bands of energy and prohibited intervals.

A rare series of black cristalline products of the charge transfer complex class derived from ethylene­dioxyethylenedlthiotetrathiafulvalene (EDOEDTTTF), ethyl­enedloxymethylenedithlotetrathiafulvalene (EDOMDTTTF) , ethyl­enedioxyvinylenedithiotetrathlafulvalene (EDOVLITTTF), methylenedlthlotetra-thiafulvalene (MDTTTF). These complex salts present interesting crystallographic properties and electrlcal activity as semiconductors, conductors and superconductors.  

In the pyrrole series the homologues yield blacks which conduct electrical current. The indole and the methylindole yield blacks which conduct electrical current. Several homologous polymers obtained from DHI are reported in the literature. Direct measurement of the electrical conductivity of the natural melanins has been undertaken from 1982. Even though the number of the melanins examined is small, the conductivity of all the “black polymers” is rather low, in the sense that they are at the limit of the isolators. Besides, it is seen that there is not a great diference between the synthetic melanins and the natural melanins. The polymers, if doped, can give materials which conduct electrical current well, while for the melanins the same operation of doping has not been made. It remains, however, to consider that the low values for conductivity of the melanins may be found in the non-optimal methods of preparation and methods of purification used. It was also observed that melanins modify slowly in alkali giving rise to two parts which are optically different. The research on the electrical activity has shown properties new to the melanins or perhaps discovered new properties of biological interest for the pigment, until now considered, among other things, biological garbage. Conductivity measurements have been carried out on skin pigment, the electrical properties of amorphous semiconductor melanosome have been studied throughout melanogenesis. The presence of long-lasting current was observed in the study of catechol-melanin in the temperature range -20⁰C and 35⁰C (Ozak, W., et al., 1987). Natural and synthetic melanins have been studied with optical absorption and their photoconductivity measured in the interval 200-700 nm. Both the photoconductivity and the optical absorption increase in the ultraviolet region while a negative photoconduction is observed with a maximum at about 500 nm. These results are in agreement with the band theory for amorphous semiconductors.

 

  Charge Transfer Complexes

  Charge transfer complexes, already recognised in the l9th century, have obtained new notoriety with the discovery of the complex obtained between tetra-cyanoquinodimethane TCNQ and tetrathiofulvalene which has a metallic type conductivity comparable or better than graphite . From a theoretical point of view, melanin would be an optimal partner of the charge transfer complex. DOPA-melanln oxidises NADH and also in part NADPH, reduces potassium ferricyanide, 2, 6-dichlorophenol, indolephenol (DCPIP), the  cytochrome C . Melanins from different sources and neuromelanins possess the same properties of “electron transfer complexes” as does the dopa-melanin. This would seem to indicate that the melanin can be reversibly oxidised by ferricyanide and reduced by NADH. Sepia and hair melanin are used to form charge transfer complexes between chlorpromazine (electron donor) and melanin (electron acceptor). Testing of the complex can be undertaken by forming pills from the components (the conductivity must be higher than that of the single parts), or in solution by conductimetric titration . The formation of the charge transfer complexes observable in solvents can be carried out, before or after.

Threshold Switching

www.organicsemiconductors.com

UV irradiation. EPR, UV, IR spectrometry and NMR can be used to establish the occurrence of formation of charge transfer complexes. Although there is a well known affinity between the melanins for organic molecules and ions  the results of the study of the charge transfer complexes, electrical and bioelectrical measurements have, been scarse, so far. For some time it has been known that melanin is a “threshold switching” amorphous semiconductor. Until recently it had been thought that these properties were only common to inorganic materials. “Threshold switching measurements of synthetic melanin show that the organic semiconductor switches to a low-resistance state in low electric fields”.

Isolation of pyrrole acids from oxidation products of sepiomelanin:

“Isolation of pyrrolic acids from the oxidation products of sepiomelanin (10 g) was carried out using 36 % hydrogen peroxide (250 ml) in acetic acid (250 ml) as the oxidizing agent (Piattelli, Fattorusso Magno, 1962) . After the reduction of excess oxidant, the solution was concentrated in vacuo to 350 ml, filtered and continuosly extracted with ether (40 hours). The solvent was removed and the residue dissolved in water (20 ml); the fIltered solution, after addition of conc. H₂SO₄ (1 ml), was subjected to countercurrent distribution (150 stages) between water and ether. Analysis of the contents of each tube by paper chromatography (ethanol - 33 % NH3 - water 80: 4: 16) gave the following results:

 

tubes 2-10: one spot Rf = 0.1

tubes 13-16: one spot Rf = 0.21

tubes 17-22: two spots Rf = 0.21 and 0.51

tubes 23-31: one spot Rf = 0.51

tubes 35 42: one spot Rf = 0.75

The evaporation of the contents of tubes 2-10 left a brown tarry residue (2.25 g), which was purified by paper chromatography (n-propanol - 33 ~ NH3 - water 60: 30: 10). The band with Rf = 0.7 thus obtained was eluted with water, and the eluate evaporated to dryness, giving a brown, partially crystalline residue (0.68 g). This was again chromatographed in n-butanol - acetic acid- water 60: 15:25, and the band with Rf = 0.56 eluted with water; paper electrophoresis (electrolytes: 005 M pyridinium formate and 0.03 M potassium dihydrogen phosphate) of this solution showed that a small amount of pyrrole-2,3-dicarboxylic acid and a larger quantity of pyrrole- 2, 3, 4, 5-tetracarboxylic acid were present. The failure to separate these acids in the preparative chromatograms, run in n­butanol - acetic acid - water 60 :15: 25 (in which their Rf values are different, but rather close), was probably due to the presence of large amounts of other oxidation products(sepiomelanic acids). 10% KCL solution (0.5 ml) was added to the remainder of the eluate (12 ml) containing the two acids; the crystalline precipitate was collected and recrystallized from water (yield: 37 mg). Ion exchange on a strongly acidic resin (Dowex 50 W) gave the free acid (31 mg), which crystallized from dioxan in colourless prisms. This compound decomposes at high temp. without a sharp m.p.; its identity with pyrrole-2, 3, 4, 5-tetracarboxylic acid was shown by its infrared spectrum, by paper chromatography (ethanol - 33 % NH3 - water 80: 4 :16:

Rf = 0.1; n-propanol - 33 % NH3 - water 60: 30 :10: Rf = 0.07; n-butanol - acetic acid - water 60 :15: 25: Rf = 0.57) and by paper electrophoresis (0.05 M pyridinium formate and 0.03 M potassium dihydrogen phosphate). The combined mother liquors, obtained after precipitation and crystallization of the salt of pyrrole- 2, 3, 4, 5-tetracarboxylic acid , were passed through a column of cation exchange resin; 58 mg of residue were obtained after evaporation to dryness. This material was further purified by paper chromatography in n-butanol - acetic acid - water 60 :15: 25. Two bands of rather close Rf values were obtained from the upper band (Rf = 0.68), an additional 30 mg of pyrrole-2, 3, 4, 5-tetra- carboxylic acid (identified as above) was isolated (total yield of pyrroie-2, 3, 4, 5-tetracarboxylic acid = 61 mg). For analysis, a sample was recrystallized from water (Found: C, 18.15 %; N, 4.69 %. C₈H₃O₂N.3H₂O requires C, 18.18 % N, 4.71 %) . A crystalline material (18 mg) was eluted from the lower band (Rf = 0.62). Paper electrophoresis showed that it was a mixture of pyrrole-2, 3, 4-tricarboxylic acid and pyrrole-2, 3, 4, 5-tetracarboxylic acid . Colourless crystals (3.2 mg) were finally isolated by electrophoresis on SS 470 paper (0.05 M pyridinium formate); this product was identified with pyrrole-2, 3, 4-tricarboxylic acid (8mg) by infrared spectroscopy, chromatography (ethanol - 33 ~ NH3 - water 80: 4 :16: Rf = 0.1; n-propanol - 33 % NH3 - water 60: 30 :10: Rf = 0.07; n-butanol - acetic acid - water 60: 15: 25:Rf = 0.5) and paper electrophoresis (migrational aptitude relative to that of acid in 0.05 M pyridinium formate: 0.59; in 0.03 M potassium dihydrogen phosphate: 0.48). The residue (450 mg) from tubes 17-31 of the countercurrent distribution was chromatographed in ethanol - 33 % NH3 -water 80: 4: 16. Two bands were obtained, the upper of which had a Rf value (0.5) identical with that of pyrrole­2, 3, 5-tricarboxylic acid . From this band, 230 mg of crude acid were isolated; crystallization from acetic acid gave 200 mg of pure pyrrole-2, 3, 5-tricarboxylic acid (yield: 2.9%). The second band yielded a small amount of another unknown product. Pyrrole-2, 3-dicarboxylic acid, present in tubes 35-42, was further purified by countercurrent distribution (50 stages) between water and ether and it was estimated that there were 100 mg present.  

 

Sepiomelanin. 

 (Link 6, Link 9, Link 12)

Among the melanins sepiomelaninic acid , the most widely studied, occurs as a Ca and Mg sal in the ink gland of Sepia officinalis The polymeric material belongs probably to the cyclodopa-melanin group  and is considered an amorphous semiconductor. Already in ancient times, the ink gland of cephalopods was recognized as being an excellent source of melanin, as well as a good tool of investigating its natural process, called melanogenesis. Several studies were made on this interesting issue some years ago: accordingly, the ink resulted to be composed of melanin granules,  further cellular constituents and degraded oligomers.( See Link 12 references 26-29

 

Aspergillin.

 

The allomelanins must include Daldinia-melanin, Ustilago-melanin , humic acids , aspergillin . This latter is particularly interesting because oxidative degradation produces the same acid which can be obtained from graphite: mellitic acid. In other words it may be the case that aspergillin is a “graphite” synthesized by a living organism. Aspergillin is a pigment which gives Aspergillus niger its characteristic dark colour. The typical coloration of the conida constitutes the fundamental distinctive character of numerous species. The appearance of the pigment in the spores is at first yellow-ish, becoming green-yellow, green-grey, brown-black. These “colours” are typical of the amorphous semiconductors. The oxygen in this synthesis plays a special role next to the iron in that the quantization of the chemical elements can lead to a gold—yellow pigment. The IR spectrum in the molecular phase is very similar to that of humic acids. The band theory may be extended to these amorphous semiconductors with polycondensed nuclei. The pigment is purified dissolving in NH₄OH 5% (solubility 1g per 1000 cc) and reprecipitated with HC1 2N, washing with H₂O, dissolved again in NH₄OH 5% reprecipitated with H₂S0₄. The dissolving in NH₄OH and the precipitation with H₂SO₄ are carried out several times. The insoluble fractions are discarded. The final product is washed with H₂0, alcol, acetone, H₂O. The wet product is dissolved in water . The solution is used for measuring the conductivity (difference between conductivity of the solution and of distilled water) both in solution and in the solid state. The mass spectrometry (MALDI-TOF-MS) was not used.

The      elementary         analysis          (Sephadex purification)                  of aspergillin gives C%=52.7 H%4,0 N%=3.4 0%=36.0 . Nitrogen may be part of a protein. Aspergillin oxidised with H₂O₂ 10% yields mellitic acid (1 g from 4 g of pigment) and oxalic acid; reduced with N. Raney it yields perylene next to hydroderivatives, phenanthrene and napthalene derivatives. These degradation products are precious elements for MALDI—TOF-MS and, in general, in mass spectrometry studies. Asperglliln can be dissolved both for measurement of the electrical conductivity (perylene itself is a good conductor if doped) and for mass spectrometry studies.

Mass spectrometry

Mass spectrometry has only recently become a precious weapon in the study of some polymers. It allows the establishment of the structure of proteins, polysaccarids and biopolymers in general. The determination of terminal groups, molecular weight, of the oligomers, direct determination of the sequence and distribution of the copolymers are performed by MS. No  results  on “ melanin molecular weight “were obtained in our BCM and BSM studies (unpublished results),Melanin do not have a  ionic or molecular mass. It also seems that MALDI-TOF-MS (Matrix Assisted Laser Desorption Ionization - Time of  flight  Mass Spectrometry) or other variants of mass spectrometry together with chemical degradation products can reach a correct analysis of some polymers, a fact not easy until today. A systematic study of the natural and synthetic melanins, applying mass spectrometry, has been underway at Padova since 1985. The possibility that in conducting polymers besides stable unpaired electrons there are positive centres associated with counteranions has not been considered For the first time it has been possible to identify the o.quinone of the dopamine, the aminochrome, the semiquinone, classical intermediates in melanogenesis. 

The structural study of tryptophane- melanins allows identification of degradation products such as toluene, ethylbenzene, styrene, indole, methylindole, phenol, cresol, methylpyrrole, indolin-2-one. These demolition products are precious elements for further structural and analytical investigation but more complexe intermediates were not seen The MALDI method has also been used for determination of the molecular weight of the melanins which appears to be very much lower that expected. The determined masses oscillate between 500 — 1,500 (sometimes with a 35,000 peak) showing that they concern mainly mixtures of oligomers, proteic materials, oxidative fission and photolysis products, simple and complex salts and clatrates. The melanogenesis of 5,6-dihydroxytryptamine (5,6-DHT) and 5,7-dihydroxytryptamine (5,7-DHT) ,  one of the most important neurotoxins derived from the metabolism of serotonine,  has been studied. 

 

Briefly :

   A system with many conjugated double bonds (Little spine), in different experimental conditions, tends to transform into black products. The chain of conjugated double bonds may be part of linear or polycondensed benzene or heterocyclic systems. The black material belongs to the organic solid state and gives a broad EPR signal in the region of 2,0005 G, being an electrical conductor if doped. It shows the phenomenon of threshold switching, good conductivity as charge transfer complexes. The colour of these materials is due to electronic transitions in materials with band structure. Interesting amorphous semiconductors ( BSM ) are pyrrole-black, acetylene-black, aniline-black All these blacks are good conductors, especially when doped. For benzene black we know linear polymers, polymers with condensed nuclei and mixed polymers. The grade of polymerization, both linear and condensed, which precedes the transformation of the molecular state  to solid state  is defined at about 30-40 double bonds.

MALDI produce,like other black particle an explosion. The fragments so obtained are useful in the study of oligomers structure .  ( Link 12 )  ( Link 21)   ( Link 22) . Peculiar is the binding capacity for gases,liquids,solids, 

 

REFERENCES

 

Collection of papers to put into the text

Works on melanins, melanogenesis and fluctuating systems

 

.

1.

 

a)      A.Quilico, I pigmenti neri animali e vegetali, Ed. Fusi, Pavia 1937.

 

b)      R.H.Thomson, Melanins, Comparative Biochemistry, Ed. M.Florkin, H.S.Mason, AP. 727 (1962)

 

 

c)      R.A.Nicolaus, Melanins, Hermann, Paris 1968.

 

d)      G.Prota, Melanins and melanogenesis, AP, (1992).

 

 

e)      P.Manzelli, M.G.Costa, Il tempo come coordinata: gli studi di Giorgio Piccardi (1895-1972). Congress Nazionale di Storia e Fondamenti della Chimica, Perugia 27 Nov. 1993. This work should be read with some criticism.

 

f)        G.A.Swan, Structure, chemistry and biosynthesis of the Melanins. In Fortschritte der chemie Organische Naturstoffe, Eds. W.Hertz, H.Grisenbach, G.W.Kirby. Vol. XXXI 522-582, Springer Verlag, Wein (1974).

 

 

g)      D.L.Fox, Animal Biochromes, University Californiana Press (1976), Berkely.

 

h)      R.A.Nicolaus, Melanine, Quaderni Accademia Pontaniana n. 4, Ed. Giannini, Napoli, 1984.

 

 

 

Reviews.

 

1). M. Thomas (1955), Modern Methods of Plant Analysis, Vol.4, pag 661-675. Eds K. Paech, M.V.Tracey, Springen Ferlag Berlin.

 

2). H.S. Mason (1955) "Melanins" Advances in Enzymology, 16, 105-184.

 

3). H.S. Mason, (1957) "Melanins" Advances in Enzymology 19, 79-233.

 

4). R.H. Thomson (1957) "Naturally Occuring Quinones" Butterworths, London.

 

5). H.S. Mason (1959) "Structure of Melanins" in Pigment cell Biology, Ed. M. Gordon AP New York Pag. 563-583.

 

6). R.H. Thomson (1962) "Melanins" 727-754, Comparative Biochemistry Vol. III, AP, New York and London.

 

7). R.H. Thomson (1962) "Some Naturally occurring black pigments" in Chemistry of natural and synthetic colouring matters and related fields AP, New York 99-113.

 

8). G.A. Swan (1963) "Chemical Structure of Melanins" 1005-1016, Annals of the New York Academy of Sciences Vol. 100.

 

9). G.A. Swan (1964) "Some Studies on the formation and Structure of Melanins" 1-20, Rend.Acc.Sci.Fis.Mat. XXXI. Napoli.

 

10). G.A. Swan (1974) "Structure, Chemistry and biosynthesis of the melanins", 522-528 in Fortschritte der Chemie Organischer Naturstoffe Vol. 31 Springer-Verlag, Wien.

 

10a). R:A.Nicolaus  ‘’ The chromatographic study of pyrrolic acids arising from oxidative degradation of natural pigments  ‘’  Rassegna di medicina sperimentale VII, 1-23, 1960.

 

10b). R.A.Nicolaus ‘’  Biogenesis of melanin  ‘’  Rassegna di medicina sperimentale IX,Suppl.1,1-32, 1962

 

10c). M.Rolland  ‘’  Oxidation des restes de tyrosine des proteines par la polyphenolxidase ‘’  These 30/XI/1962 n° 108, Facultè des Sciences, Universitè D’Aix-Marseille.

 

10d). R.A.Nicolaus,M.Piattelli,  ‘’ Progress in the chemistry of natural black pigments ‘’  Rend.Acc.Sci.Fis.Mat., XXXII, 1-17, 1965.

 

10e). R.A.Nicolaus  ‘’  Chimica delle melanine naturali  ‘’  ( Conferenza alla Sezione Lombarda della Società Chimica Italiana, Milano 27 Ottobre,1965 ) , Chimica ed Industria, 48, 341-347, 1966.

 

.

 

Recent reviews on melanin chemistry

 

11). G. Prota (1988) "Progress in the Chemistry of Melanins and related metabolites" Medicinal Research Reviews 8, 525-556.

 

12). G. Prota (1988) "Some new aspects of melanin chemistry" Advances in pigment Cell Research, (J.T. Bagnara Ed), 101-124, A. Liss, New York.

 

13). G. Prota (1989) "Melanins and pigmentation Coenzymes and cofactors" (D. Dolphin R.Paulson, O. Abramovic eds) Vol 3, 441-466, J. Wiley, New York.

 

14). G. Prota (1992) "Melanins and Melanogenesis" AP, San Diego pp.1-290

 

15). G. Prota (1993) "Melanins and related metabolites" in Black Skin, W. Montagna, G. Prota, J.Kenney, pp. 73-99

 

16). G. Prota (1995) "The Chemistry of melanins and Melanogenesis" Prog. Chem. Org. Nat. Prod. Eds. W. Herz, G.W. Kirby, R.E. Moore, W. Steglich, Ch. Tamm. pp 94-148 Springer- Verlag, Wien and New York.

 

17). R.A. Nicolaus, G. Scherillo (1995) "La Melanina un riesame su struttura, propietà e sistemi" Atti Accademia Pontaniana Vol. XLIV pp. 265-287.

 

18). B.J. Nicolaus, R.A. Nicolaus (1996) "Speculating on the Band Colours in Nature" Atti Accademia Pontaniana Vol. XLV pp. 365-385

 

19). R.A. Nicolaus (1997) "Coloured organic semiconductors: melanins" Rend.Acc.Sci. Fis. Napoli LXIV, 325-360

 

20). P.A. Riley (1997) "Melanin" Int.J.Biochem, Cell Biol. 29, 1235-1239.

 

21). P.A. Riley (1997) "Epidermal melanin: Sun screen or waste disposal ?" Biologist 44, 408-4.

 

22). G. Nicolaus, R. A. Nicolaus (1999) "Melanins, Cosmoids, Fullerenes" Rend. Acc. Sci. Fis. Mat. Napoli Vol. LXVI, 131- 158.

 

23). G. Prota (2000) "Melanins, Melanogenesis and melanocytes: Looking at their functional significance from the Chemist's viewpoint" Pigment Cell Res. 13 283-293

A social event of melanin Chemistry.

 

24). R.A. Nicolaus, G. Parisi (2000) "The Nature of Animal Blacks" Atti Accademia Pontaniana, Napoli Vol. XLIX, 197-233.

 

25). A. Bolognese, R.A. Nicolaus (2001) "Natural Black Conductors" Atti Accademia Pontaniana, Vol. L, 210- 224.

 

26).  http://www.tightrope.it/nicolaus/index.htm

 

            27).  http://www.organicsemiconductors.com  

 

            28). R.A.Nicolaus  ‘’  Le melanine del cosmo  ‘’  Rend.Acc.Sci.Fis. LXIV, 1-2, 1999

 

29). B.J.R.Nicolaus, R.A.Nicolaus, M.Olivieri  ‘’  Riflessioni sulla materia nera interstellare ‘’ Rend.Acc.Sci.Fis.Mat. LXVI,110-129, 1999.

 

30). B.J.R.Nicolaus, R.A.Nicolaus  ‘’  Lo scrigno oscuro della vita  ‘’ Atti Accademia Pontaniana , XLVIII, 355-380, 2000.

 

31). R.A.Nicolaus ‘’  Divagazioni sulla struttura a bande del colore in natura  : il nero  ‘’  Rend.Acc.Sci.Fis.Mat.  LXIV, 145-216, 1997.

 

32). R.A.Nicolaus ‘’  Maldi mass spectrometry and melanins  ‘’  Rend.Acc.Sci.Fis.Mat. LXIV, 315-324, 1997.

 

33). U.Mars  ‘’  Melanogenesis as the basis for melanoma targeting ‘’   1-48, AUU,,Uppsala 1998.

 

34). R.A.Nicolaus  ‘’  The chemistry of interstellar black matter  ‘’  Atti Accademia Pontaniana, CB, XLVIII, 486-42000

 

35). R.A.Nicolaus ‘’ Le melanine.Soluzione dell’enigma chimico : prospettive in biologia’’  Atti Accademia Pontaniana CB, XLIX, 304-306, 2001.   

 

 

36). A.Bolognese, R.A.Nicolaus  ‘’  About the structure of sepiomelanin  ‘’  Atti Accademia Pontaniana CB, 309-312 , 2001.

 

37). A.Bolognese,R.A.Nicolaus ‘’  Nero di Adrenalina  ‘’  Atti Accademia Pontaniana CB, L, 391-393, 2002.

 

38). A.Bolognese,R.A.Nicolaus  ‘’  Conduttori biologici neri  ‘’  Atti Accademia Pontaniana CB,

393-394, 2002

 

39). R.A.Nicolaus,A.Bolognese, B.Nicolaus  ‘’  The pigment cell and its biogenesis  ‘’  Atti Accademia Pontaniana,  L, 225-243, 2002.

 

Chemical studies on natural melanins and in particular on sepiomelanin.

 

 

2

 

a)      L.Panizzi, R.Nicolaus - Ricerche sulle melanine. I. Sulla melanina di seppia. Report of Scienze Fisiche, Matematiche e Naturali della Accademia dei Lincei, Serie VIII, Vol. XII, 420 (1952)

b)      L.Panizzi, R.Nicolaus - Ricerche sulle melanine. Nota I. Sulla melanina di seppia. Gazz. Chem. Ital., 82, 435 (1952);

c)       M.Piattelli, R.A.Nicolaus - The structure of melanins and melanogenesis. I. The structure of melanin in Sepia. Tetrahedron, 15, 66 (1961);

d)      M.Piattelli, E.Fattorusso, S.Magno, R.A.Nicolaus - The structure of melanins and melanogenesis. II. Sepiomelanin and synthetic pigments. Tetrahedron, Vol. 18, 941 (1962);

e)      M.Piattelli, E.Fattorusso, S.Magno, R.A.Nicolaus - The structure of melanins and melanogenesis. III. The structure of sepiomelanin. Tetrahedron, Vol. 19, 2061 (1963)

f) M.Piattelli, E.Fattorusso, S.Magno, R.A.Nicolaus - The structure of melanins and melanogenesis. IV. On some natural melanins. Ibidem, Vol. 20, 1136 (1964).

 

g) E.Fattorusso, M.Piattelli, R.A.Nicolaus, S.Magno - The structure of melanins and melanogenesis. V. Ustilago-melanin. Tetrahedron, Vol. 21, 3229 (1965);

 

 h). R.A.Nicolaus - Biogenesis of melanins. Rassegna di Medicina Sperimentale, Anno IX, Supplemento N°1, Ed. V.Idelson, Napoli (1962).

 

i)  M.Benathan - Contribution à l’analyse quantitative des mèlanines. Application comparée de mètodes de caractérisation et de degradaation oxidative à la melanine de l’encre de seiche, à la melanine de l’iris de boeuf et à la dopa-melanine. PhD thesis presented to the Faculté des Sciences de l’Université de Lausanne, Thesis supervisor Prof. H.Wyler, Imprivite S.A., Lausanne 1980.

 

Pyrrole- black

 

3

 

a)      A.Dall’Olio, G.D’Ascola, V.Varacca, V.Bocchi, Resonance paramagnetique electronique et conductivité d’un noir d’oxypyrrole électrolytique, presentée par M. René Lucas, Comptes Rendues Academie Scientifique, Paris, 267 433 (1968).

 

b)      S.Jasne in Encyclopedia of Polymer Science and Engineering, II Edition, Vol. 13 Polypyrroles 42 (1985).

 

 

c)      G.P.Gardini, A.Berlin, I polimeri conduttori, Chimica e Industria 73 764 (1991).

 

d)      A.Pullman, B.Pullman, The Band structure of Melanins, Biochim. Biophys. Acta 54 384 (1961).

 

 

Humic acids and melanins produced by microrganisms

 

 

4

 

.

a). M.M.Kononova, Soil Organic Matter, Permagon Press 1961.

 

b). M.H.B.Hayes, P.MacCarty, R.L.MacColm, R.S.Swift, Humic Substances, Wiley 1989.

 

 

c). S.P.Liach, E.L.Ruban, Microbnye Melaniny, Akad. Nauk. SSSR, Institut Mikzobiologii, Izd. Nauka 1-86, Moskova 1972.

 

d). A.A.Bell et al., Tetrahedron 32 1353 (1976).

 

e). Ciny Lee et al., Organic Matter in Sea Water Biogeochemical Processes, Chemical Oceanographiic, V. 9 1-49 (1989), Ed. Riley, Accademic Press.

 

 

Various melanogens

 

Substances able to give black or brown composites by chemical or enzymatic oxidation are called melanogens. A melanogen can produces red-brown, red, orange, yellow and rarely green "pigment". The pigments have an EPR signal and are all potential electrical and sound conductors.

 

5.

 

a)      S.Roy, A.K.Chakraborty, .D.P.Chakraborty, Melanin formation and breakdown of Indole under Underfriend conditions, J. Chem. Soc. 58 992 (1981).

 

b)      G.Cimino, S.De Rosa, S.De Stefano, A.Spinella, G.Sodano, The Zoochrome of the Sponge Verongia Aerophoba (Uranidine), Tetrahedron Letters 25 2925 (1984).

 

 

c)      G.Natta, G.Mazzanti, P.Corradini, Polimerizzazione stereospecifica dell’acetilene, Rendiconti Accademia dei Lincei, XXV (1958); J.D.Bu’Lock, Quart. Revs. (London), 10 371 (1956).

 

d)      L.Horner, K.Sturm, Modellreaktion zur Melaninbildung, Liebigs Ann. Chem. 608 128 (1957).

 

 

e)      B.Schmidli, Über Melanine, die dunklen Haut und Haarpigmente, Helv. Chim. Acta XXXVIII 1078 (1955). (prepares melanin from hystidine, hystamine, carnpsine, DOPA; tyrosine, phenol, phenylalanine, pyrrol, tyramine and melanin from hair. Spearation by column chromatography)

.

f)        P.A.Wehrli, F.Pigott, U.Fischer, A.Kaiser, Oxidations produkte von 6-hydroxy-dopamine, Helv. Chim. Acta LV 3057 (1972); R.J.S.Beer, T.Broadhurst, A.Robertson, The Chemistry of Melanins. Part V. The autoxidation of 5,6-dihydroxyindoles, J. Chem. Soc. 1947 (1954).

 

g)      The melanogenetic properties of tryptophane and its derivatives have been the object of extensive studies by L.Musajo and his School of Padova. Some works which concern this partial literature are presented here.

 

 

h)      E.M.Nicholls, K.Rienits, Marsupial pigpents, in Pigment Cell, Mechanism in Pigmentation, Vol. 1, pag. 142. Ed. McGovern, Russel, Riley S.Karger Basel 1973; A.Bolognese, C.Piscitelli,

i)         G.Scherillo, Formation of dihydro and dihydroisotriphenodioxazines by acidic treatment of some substituted 3H-phenoxazin-3-ones: isolation and characterization. A new perspective in the chemistrry of ommochromes. J. Org. Chem., 48, 3649 (1983); A.Bolognese, R.Liberatore, G.Riente, G.Schirillo, Oxidation of 3-hydroxykynurenine. A reexamination. J. Heterocyclic Chem., 25, 1247 (1988).

       m) T.Uemura, T.Shimazu, R.Miura, T.Yamano, NADPH dependent melanin pigment formation from 5-hydroxyindolealkalamines by hepatic and cerebral microsomes, Biochem. Biophys. Res. Comm. 93 1074 (1980).

 

n) F.Celentano, Luce, colore e materia, Le Scienze n. 21, Feb 1985, Milano 1985.

m) J.Itten, Arte e colore, Il Saggiatore, Milano 1965.

 

o) Green is rarely represented. We may recall emeraldine chlodhydrate (3, c) and biverdina (B.Pullman, M.Perault, Proc. Nat. Acad. Sci. U.S. 45 1476 (1959).

 

Radicals and semiconductors

 

 

 6

 

H.C.Longuet-Higgins, On the origin of the free radical properties of melanins, Arch. Biochem. Biophys. 86 225 (1960);

 

 M.Brindelli, R.Cappelletti, P.R.Crippa, Electret state and hydrated structure of melanin, Biol. Biochem. 8 555 (1981);

 

 H.S.Mason, J. Biol. Chem. 172 83 (1948).

 

J.E.McGinness, Mobility gaps: a mechanism for band gaps in melanin, Science 177 896 (1972);

 J.E.McGinness, P.Proctor, The importance of the fact that melanin is black, J. Theor. Biol. 39 677 (1973); J.E.McGinness, P.M.Corry, P.Proctor, Amorphous semiconductor switching in melanins, Science 183 853 (1974)

 

 T.Sarna et al., Biophys. J., 16 1165 (1976).

 

  W.A.Waters, J. Chem. Soc. (B) 2026 (1971);

 

 G.A.Swan, An Introduction to the alkaloids, Blackwell Scientific Publications, Oxford and Edinburg 1967;

 

 D.H.R.Barton, Proc. Chem.Soc. 293 (1963); R.H.I.Manske, The Alkaloids, AP, New York 1960.

M.R.Chedekel et al., J. Chem. Soc. Chem. Comm. 1170 (1984).

 

 D.S.Galvano, M.J.Caldas, Polymerization of 5,6-indolequinone: a view into the band structure of melanins, J. Chem. Phys. 88 4088 (1988); J. Chem. Phys. 93 2848 (1990); J. Chem. Phys. 92 2630 (1990).

 

 

 C.H.Culp, D.E.Eckels, P.H.Sidles, Threshold switching in melanin, J. Appl. Phys. 46 3658 (1975).

 

J.E.Wertz, D.C.Reitz, F.Dravnieks, Electron Spin Resonances Studies of autoxidation of 3,4-dihydroxyphenyl alanine, in M.S.Blois, H.W.Brown, R.M.Lemmo,, R.O.Lindblom, M.Weiss Bluth, Free Radicals in Biological Systems, AP 1961.

 

 T.J.Stone, W.A.Waters, J. Chem. Soc. 213 (1964);

 

  A.R.Forrester, J.M.Hay, R.H.Thomson, Organic Chemistry of Stable Free Radicals, pp.280-405,AP, London New York 1968.

 

L.Zanotti, G.Aureli, M.Rizzotti, G.Succi - Misure di risonanza paramagnetica elettronica su peli di bovini a diverso mantello. Ibidem, Vol. XIII, 1967 (Milano);

p). M.Rizzotti, R.Striani Pannelli, G.Aureli, L.Zanotti, G.Succi - Ricerche morfologiche e istochimiche sulla pelle e biofisiche sui peli di bovini albini. Ibidem, Vol. XV, 1969 (Milano); H.U.Winzenreid, J.J.Lauvergne, Spontanes Suftreten von Albinos in der Schweizerischen Braunviehrasse, Schweizer Archiv. für Tierheilkunde, 112 581 (1970).

 

 M.A.Pathak, in Advances in Biology of skin, Vol. VII, Ed. W.Montagna, Permagon Press, Oxford and New York 1967.

 

 

R.A.Nicolaus, Biogenesis of melanins. Rassegna di Medicina Sperimentale, Anno IX, Supplemento N°1, Ed. V.Idelson, Napoli (1962).

 

K.Nassau, in Le Scienze quaderni n. 21 February 1985, 59, Milano, 1984.

 

 

B.T.Allen, D.J.E.Ingram, The Investigation of Unpaired Electron Concentration produced in large Molecules by ultraviolet Irradiation, in Free Radicals in Biological Systems 218, Ed. M.S.Blois, H.W.Brown, R.M.Lemmon, R.O.Lindborn, M.Weissbluth, AP (1961).

 

A.Bolognese, R.Bonomi, R.Chillemi, S.Sciuto, Oxidation of 3-hydroxykynurenine. An EPR investigation, J. Heterocyclic Chem., 27, 2207 (1990).

 

 

R.Seraglia, P.Traldi, G.Elli, A.Bertazzo, C.Costa, G.Allegri, Laser desorption ionization Mass Spectrometry in the study of Natural and Synthetic Melanins. 1. Tyrosine Melanins, Biol. Mass Spectrom. 22, 687 (1993).

 

W.A.Little, Possibility of synthesizing an organic superconductor, Physical Review 134, A116 (1964).

 

 

       LINK 22

 

Appendice

1950-2000 Conductivity and related papers relevant for pigment cell research.

 

 

1) F. London, 1950 "Superfluids", vol. 1, J. Wiley, New York.

2) Darrow, K.K., (1954). "I semiconduttori". Endeavour XIII, N. 50, pp. 101-106.

3) Commoner, B., Townsend. J., Pake, G., (1954) "Free radicals in biological materials" Nature 174,689.

4) W.G. Walter (1954) "The electrical activity of the brain" Scientific American, Published by W.H. Freeman, 660 Market Street, San Francisco 4, California, USA.

5) Mason, H.S., Fowlks, W.L., Peterson, E., (1995). "Oxygen transfer and electron transport by the phenolase complex". J. Am. Soc. 77, 2914-2915.

6) Keynes, R.D., (1956). "La produzione di elettricità nei pesci". Endeavour N. 60 pag.215-230.

7) N. Millott (1957) "La fotosensibilità animale particolarmente nelle forme prive degli occhi" Endeavour, Vol. XVI, 19-29.

8) J. Bardeen, L. N. Cooper, J. R. Schrieffer, (1957) Phys. Rev., 108, 1175.

9) A. Pullman, B. Pullman "The Band Structure of Melanins" Biochim. Biophyis. Acta 54, 384 (1961); B. Pullman, A. M. Perault, Proc. Natl. Acad. Sci., USA, 45, 1496/1497 (1959).

10) N.B. Hannay (1959) "Semiconductor" pag. 551, Rheinold, New York.

11) K.Herzfeld, T.A Lictowitz (1959) "Absorption and dispersion of ultrasonic waves" AP, New York.

12) P.George, J.S. Griffith (1960) "Electron trasfer and Enzyme Catalysis" John Harrison Laboratory of Chemistry, University of Pennsylvania, Philadelphia, and Department of Theoretical Chemistry, The University of Cambridge, Cambridge, England.

13) Mason, H.S., Ingram, D.J.E; Allen, B., (1960). "The free radical property of melanins". Arch. Biochem. Biophys. 83, 225.

14) Longuet-Higgins, H.C., (1960). "On the origin of the free radical properties of melanins". Arch. Biochem. Biophys. 86, 231-232.

15) R.O. Becker (1961) "Search for evidence of Axial Current Flow in Peripheral Nerves of Salamander" Science, 134, 101-102.

16) E.P. Wigner, "The logic of personal knowledge", pag. 231, (Rouledge Routledge and Kegan Paul, London 1961).

17) Allen, B.T., Ingram, D.J.E., (1961) "The investigation of the umpaired electron concentrations produced in large molecules by ultraviolet irradiation" in Blois, Brown, Lemmon, Lindblom, Weinbluth "Free Radicals in biological systems" 211, AP, New York.

19) R. Fujii, (1961) "Demonstration of the Andrenergic Nature of Trasmission of the junction between Melanophore-concentrating nerve and Melanophore in Bony Fish", J. Faculty of Science 9, 171-196. University of Tokio.

20) E.A. Lynton (1962) "Supercondutivity" J. Wiley New York.

21) Cope, F.W., Sever, R.J., Polis, B.D., (1963) "Reversible free radical generation in the melanin granules of the eye by visible light" Arch. Biochem. Biophys. 100, 171-177.

22) B. Pullmann (1963) "Sur la biogenese des melanins" BBA, 66, 164-165.

23) W.A. Little (1964) "Possibility of synthesing an organic superconductor" Phys. Rev. 134, A1417.

24) J.E.Kurler (1964) "I magneti superconduttori" Endeavour XXIII, 115-121.

25) P. Seybold, M. Gouterman (1964) "Radiation Transitions in Gases and Liquids" 413-433, Conant Chemical Laboratory, Harvard University, Cambridge Massachusetts 02138.

26) Parks, R.D., (1965). "Quantum effects in superconductors". Scientific American, 1965 pp. 57-67.

27) Blois, M.S., (1965). "Random polymers as a matrix for chemical evolution". In: The origins of Prebiological systems, ed. Fox, S.W., AP, New York pp.19-39.

28) Bassett, C.A.L., (1965). "Electrical effects in bone". Scientific American, pp. 18-26.

29) Bowen, E.J., (1965). "Recent progress in Photobiology". Blackwell Oxford.

30) Little, W.A., (1965) "Superconductivity at Room Temperature" Scientific American 212, pp. 21-27.

31) G. Tollin, C. Steelink. (1966) "Biological Polymers Related to catechol: electron paramagnetic resonance and infrared studies of melanin, tannin, lignin humic acid and hydroxyquinone" BBA 112, 377-379.

32) Bolt, A.G., Forrest, I.S., (1966) "Charge-transfer reaction between melanin and chloropromazine" Paper given at 152 nd ACS Meetting, New York City.

33) Rabenau, A., (1966), "I problemi chimici negli studi sui semiconduttori". Endeavour XXV, pp. 158-165.

34) Pathak, M.A., (1967). "Photobiology of melanogenesis: Biophysical aspects". In Advances in Biology of Skin (W. Montagna and F. Hu, eds.), Vol. 8, pp. 397-420. Pergamon Press, Oxford.

35) Mulay, I.L., Mulay, L.N., (1967). "Magnetic susceptibility and electron spin resonance absorption spectra of mouse melanomas S91and S91A". J. Natn. Cancer Inst. 39, 735.

36) F.Gutman, L.E. Lyon (1967) "Organic Semiconductors" Wiley, New York

37) Simmons, J.E., (1968) "Electrical conduction in thin insulating films" Endeavour XXVII, N. 102, pp 138-143.

38) Pathak, M.A., Stratton, K., (1968). "Free radicals in human skin before and after exposure to light" Archiv. Biochem. Biophys. 123, 468-476.

39) Dall'Olio, A., Dascola, G., Varacca, V., Bocchi, V., 1968 "Resonance paramagnetique elettronique et conductivité d'un noir d'oxypyrrol electrolytique", C.R. Acad. Paris. 267, 433-435.

40) Forrester, A.R., Hay, J.M., Thomson, R.H., (1968). "Organic Chemistry of stable Free Radicals". AP, London p. 1-405.

41) Y.T. Thathachari, M.S. Blois (1969). "Physical Studies on Melanins. Biophysical Journal, Vol. 9, 77

42) Warren, B.E., (1969) "X-rays difraction". Addison-Wesley, New York.

43) Blois, M.S., (1969). "Recent developments in the physics and chemistry of the melanins". In: The biologic effects of ultraviolet Radiation . Ed. Urbach, F., Pergamon Press, Oxford p. 299.

44) Davis, E.A., Mott,N.F., (1970) "Conduction in non-crystalline Systems. V. Conductivity, Optical Absorption and Photoconductivity in Amorphous Semiconductors" Philos. Magazine, 22, 903.

45) Trukhan, E.M., Perveozchikov, N.F., Ostrovskii, M.A., (1970) "Biophysics", 15, 1090; 18, 413 (1973).

46) B.Gross (1971) "The Electret" Endeavour Vol. XXX, 115-119.

47) G.P. Gardini (1973) "The oxidation monocyclic pyrrole" in Advances in Heterocyclic Chemistry, Ed.A.R. Katritzky, A.J. Boulton Vol. 15, 67-98, AP New York.

48) Coleman, L.B., Cohen, M.J., Sandam, D.J., Yamagishi, F.G., Garito, A.F., Heeger, A.J., (1973), Solid State Comm. 12, 1125.

49) R.B. Stephens, Phys. Rev. B; 8 2896 (1973); T. Egami, O.A. Sacli, A.W. Simpson, A.L. Terry "Amorphous Magnetism" pag. 27-45 in H.O. Hooper, A.M. deGraaf, Plenum Press, New York 1973.

50) McGinness, J.E., Corry, P,P., Proctor, P., (1974). "Amorphous semiconductor switching in melanins". Science 183, 853-854.

51) J.E. McGinness "Mobility gaps: a mechanism for band gaps in melanin". Science, Vol. 177, 896 (1972); J.E. McGinness, P. Proctor "The importance of the fact that melanin is black". J. Theor. Biol., 39, 677 (1973).

52) P.S. Farago (1974) "Polarized electrons" Endeavour XXXIII.

53) Goodings, E.P., (1975). "Polymeric conductors and superconductors". Endeavour XXXIV, N. 123, September, pp. 123-130.

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The chemistry of evolution

 

 

7

 

.

a)      J.D.Bernal, Molecular Matricies for living Systems, in the Origins of Prebiological Systems and their Molecular Matrices, Ed. S.W.Fox, AP 1965; M.S.Blois, Random polymers as a Matrix for chemical evolution, Idem, AP 1965.

 

Melanogenesis in mammals

 

 

8.

 

 

a)      P.Azoca, F.Solano, C.Salinas, J.C.Garcia-Borron, J.A.Lozano, Regulation of the final phase of mammalian melanogenesis: the role of dopachrome tautomerase and the ratio beuween 5,6-dihydroxyindole-2-carboxylic acid and 5,6-dihydroxyindole, Eur. J. Biochem. 208 155 (1992).

 

b)      T.Kobayashi, K.Urabe, A.Winder, C.Jiminez-Cervantes, G.Imokawa, T.Brewington, F.Solano, J.C.Garcia-Borron, V.J.Hearing, Tyrosinase related protein 1 (TRP 1) functions as a DHICA oxidase in melanin biosynthesis, The EMBO Journal 13 n. 24, 5818 (1994)

 

.

c). LINK 22

 

 

 

 

The full italian version of this paper with figures, R.A.Nicolaus,G.Scherillo '' La Melanina.Un riesame su struttura,proprietà e sistemi '' Atti della Accademia Pontaniana,Vol.XLIV,265-287,Napoli 1995,is available on request.

 

Terms used

BCM =  black cell matter

BSM = black synthetic matter

granule =  melanosome whithout enzymatic or chemical activity

dopachrome = a red solution obtained from tyrosine containing quinones and phenols  

Revised September 2003